1. Field of the Invention
This invention relates generally to a mutant factor VIII having increased half-life, methods of production, pharmaceutically acceptable compositions and uses thereof. This invention also relates to a method of using receptor associated protein to increase the half-life of factor VIII, methods of production, pharmaceutically acceptable compositions and uses thereof.
2. Related Art
Coagulation of blood occurs by either the “intrinsic pathway” or the “extrinsic pathway,” whereby certain blood proteins interact in a cascade of proteolytic activations to ultimately convert soluble fibrinogen to insoluble fibrin. These threads of fibrin are cross-linked to form the scaffolding of a clot; without fibrin formation, coagulation cannot occur.
The intrinsic pathway consists of seven steps: (1) the proteolytic activation of factor XII; (2) activated factor XII cleaves factor XI to activate it; (3) activated factor XI cleaves factor IX, thereby activating it; (4) activated factor IX interacts with activated factor VIII to cleave and activate factor X; (5) activated factor X binds to activated factor V on a membrane surface, which complex proteolytically cleaves prothrombin to form thrombin; (6) thrombin proteolytically cleaves fibrinogen to form fibrin; (7) fibrin monomers assemble into fibrils, which are then cross-linked by factor XIII.
The extrinsic pathway consists of the following steps: (1) upon rupture of a blood vessel, factor VII binds to tissue factor, a lipoprotein present in tissues outside the vascular system; (2) factor VII is activated to factor VIIa by proteolytic cleavage; and (3) the factor VIIa-tissue factor complex cleaves and activates factor X. Thereafter, the extrinsic pathway is identical to the intrinsic pathway, i.e. the two pathways share the last three steps described above.
The plasma glycoprotein factor VIII circulates as an inactive precursor in blood, bound tightly and non-covalently to von Willebrand factor. Factor VIII (fVIII) is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor (vWf) and activates its procoagulant function in the cascade. In its active form, factor VIIIa (fVIIIa) functions as a cofactor for the factor X activation enzyme complex in the intrinsic pathway of blood coagulation, and it is decreased or nonfunctional in patients with hemophilia A.
In hemophilia, blood coagulation is impaired by a deficiency in certain plasma blood coagulation factors. People with deficiencies in factor VIII or with antibodies against factor VIII suffer uncontrolled internal bleeding that may cause a range of serious symptoms unless they are treated with factor VIII. Symptoms range from inflammatory reactions in joints to early death. The classic definition of factor VIII, in fact, is that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A. A deficiency in vWf can also cause phenotypic hemophilia A because vWf is an essential component of functional factor VIII. In these cases, the half-life of factor VIII is decreased to such an extent that it can no longer perform its particular functions in blood-clotting.
The fVIII protein consists of a homologous A and C domains and a unique B domain which are arranged in the order A1-A2-B-A3-C1-C2 (Vehar, G. A., et al., Nature 312:337-340 (1984)). It is processed to a series of Me2+ linked heterodimers produced by cleavage at the B-A3 junction (Fay, P. J., et al., Biochem. Biophys. Acta. 871:268-278 (1986)), generating a light chain (LCh) consisting of an acidic region (AR) and A3, C1, and C2 domains and a heavy chain (HCh) which consists of the A1, A2, and B domains (FIG. 1).
Activation of fVIII by thrombin leads to dissociation of activated fVIII (fVIIIa) from vWf and at least a 100-fold increase of the cofactor activity. The fVIIIa is a A1/A2/A3-C1-C2 heterotrimer (Fay, P. J., et al., J. Biol. Chem 266:8957-8962 (1991)) in which domains A1 and A3 retain the metal ion linkage (FIG. 1) and the stable dimer A1/A3-C1-C2 is weakly associated with the A2 subunit through electrostatic forces (Fay, P. J., et al., J. Biol. Chem 266:8957-8962 (1991)). Spontaneous dissociation of the A2 subunit from the heterotrimer results in non-proteolytic inactivation of fVIIIa.
The A2 domain is necessary for the procoagulant activity of the factor VIII molecule. Studies show that porcine factor VIII has six-fold greater procoagulant activity than human factor VIII (Lollar, P., and E. T. Parker 266 J. Biol. Chem. 12481-12486 (1991)), and that the difference in coagulant activity between human and porcine factor VIII appears to be based on a difference in amino acid sequence between one or more residues in the human and porcine A2 domains (Lollar, P., et al., 267 J. Biol. Chem. 23652-23657 (1992)).
Infusion of fVIII/vWf complex or purified plasma or recombinant fVIII into patients with severe hemophilia A who do not have fVIII (Fijnvandraat, K., et al., Thromb. Haemostas. 77:298-302 (1997); Morfini, M., et al., Thromb. Haemostas. 68:433-435 (1992)) or in normal individuals (Over, J., et al., J. Clin. Invest. 62:223-234 (1978)) results in a similar fVIII disappearance with a half-life of 12-14 hours. Although the complex between fVIII and vWf is crucial for normal half-life and level of factor VIII in the circulation, the mechanisms associated with turnover of fVIII/vWf complex are not well defined.
The human factor VIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337 (1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), and the amino acid sequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199, disclose a recombinant DNA method for producing factor VIII in mammalian host cells and purification of human factor VIII. Human factor VIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells) has been reported. Human factor VIII has been modified to delete part or all of the B domain (U.S. Pat. No. 4,868,112), and replacement of the human factor VIII B domain with the human factor V B domain has been attempted (U.S. Pat. No. 5,004,803). The cDNA sequence encoding human factor VIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2, respectively.
U.S. Pat. No. 5,859,204, Lollar, J. S., describes mutants of human factor VIII having reduced antigenicity and reduced immunoreactivity.
Porcine factor VIII has been isolated and purified from plasma (Fass, D. N., et al., Blood 59:594 (1982)). Partial amino acid sequence of porcine factor VIII corresponding to portions of the N-terminal light chain sequence having homology to ceruloplasmin and coagulation factor V and largely incorrectly located were described by Church, et al., Proc. Natl. Acad. Sci. USA 81:6934 (1984). Toole, J. J., et al., Nature 312:342-347 (1984) described the partial sequencing of the N-terminal end of four amino acid fragments of porcine factor VIII but did not characterize the fragments as to their positions in the factor VIII molecule. The amino acid sequence of the B and part of the A2 domains of porcine factor VIII were reported by Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). The cDNA sequence encoding the complete A2 domain of porcine factor VIII and predicted amino acid sequence and hybrid human/porcine factor VIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine factor VIII and a chimeric factor VIII with porcine A1 and/or A2 domains substituted for the corresponding human domains were reported in WO 94/11503. U.S. Pat. No. 5,859,204, Lollar, J. S., discloses the porcine cDNA and deduced amino acid sequences.
Cellular endocytosis mediated by LRP was shown to be a mechanism of removal of a number of structurally unrelated ligands including several proteins related to coagulation or fibrilolysis. These ligands are: complexes of thrombin with antithrombin III (ATIII), heparin cofactor II (HC11) (Kounnas, M. Z., et al., J. Biol. Chem. 271:6523-6529 (1996)), protease nexin I (Knauer, M. F., et al., J. Biol. Chem. 272:12261-12264 (1997)), complexes of urokinase-type and tissue-type plasminogen activators (u-PA and t-PA, respectively) with plasminogen activator inhibitor (PAI-1) (Nykjaer, A., et al., J. Biol. Chem. 267:14543-14546 (1992); Orth, K., et al., Proc. Natl. Acad. Sci. 89:7422-7426 (1992)), thrombospondin (Mikhailenko, I., et al., J. Biol. Chem. 272:6784-6791 (1997)), tissue factor pathway inhibitor (TFPI) (Warshawsky, I., et al., Proc. Natl. Acad. Sci. 91:6664-6668 (1994)), and factor Xa (Narita, M., et al., Blood 91:555-560 (1998); Ho, G., et al., J. Biol. Chem 271:9497-9502 (1996)).
LRP, a large cell-surface glycoprotein identical to α2-macroglobulin receptor (Strickland, D. K., et al., J. Biol. Chem. 265:17401-17404 (1990)), is a member of the low density lipoprotein (LDL) receptor family which also includes the LDL receptor, very low density lipoprotein (VLDL) receptor, vitellogenin receptor and glycoprotein 330 receptor. LRP receptor consists of the non-covalently linked 515 kDa α-chain (Herz, J., et al., EMBO J. 7:4119-4127 (1988)) containing binding sites for LRP ligands, and the 85 kDa transmembrane β-chain. Within the α-chain, cluster of cysteine-rich class A repeats is responsible for ligand binding (Moestrup, S. K., et al., J. Biol. Chem 268:13691-13696 (1993)). In contrast to the acidic ligand binding region in LRP, ligands of LRP expose regions rich in positively charged amino acid residues (Moestrup, S. K., Biochim. Biophys. Acta 1197:197-213 (1994)). This type of binding and 31 class A repeats present in LRP may be responsible for its wide ligand diversity and ability to serve as a multi-ligand clearance receptor. LRP is expressed in many cell types and tissues including placenta, lung and brain (Moestrup, S. K., et al., Cell Tissue Res. 269:375-382 (1992)) and is a major endocytic receptor in the liver (Strickland, D. K., et al., FASEB J. 9:890-898 (1995)).
A 39 kDa receptor-associated protein (RAP) binds to LRP with high affinity (Kd=4 nM (27)) and inhibits binding and LRP-mediated internalization and degradation of all ligands (Moestrup, S. K., Biochim. Biophys. Acta 1197:197-213 (1994); Williams, S. E., et al., J. Biol. Chem. 267:9035-9040 (1992)), therefore serving as a useful tool for testing whether LRP is involved in endocytosis of a given ligand.
Severe hemophiliacs, who number about 10,000 in the United States, can be treated with infusion of human factor VIII, vWf/factor VIII complex or vWf which will restore the blood's normal clotting ability if administered with sufficient frequency and concentration. However, supplies have been inadequate and problems in therapeutic use occur due to difficulty in isolation and purification, immunogenicity, and the necessity of removing the AIDS and hepatitis infectivity risk.
Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These include a partially-purified factor VIII derived from the pooled blood of many donors that is heat- and detergent-treated for viruses but contains a significant level of antigenic proteins; a monoclonal antibody-purified factor VIII that has lower levels of antigenic impurities and viral contamination; and recombinant human factor VIII, clinical trials for which are underway. Unfortunately, human factor VIII is unstable at physiologic concentrations and pH, is present in blood at an extremely low concentration (0.2 μg/ml plasma), and has low specific clotting activity.
The problems associated with the commonly used, commercially available, plasma-derived factor VIII have stimulated significant interest in the development of a better factor VIII product. There is a need for a more potent factor VIII; a factor VIII that is stable at a selected pH and physiologic concentration; a factor VIII that is has a longer half-life in circulating blood.